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Natural compounds that pose no significant medical or environmental side effects are potential sources of antifungal agents, either in their nascent form or as structural backbones for more effective derivatives. Kojic acid (KA) is one such compound. It is a natural by-product of fungal fermentation commonly employed by food and cosmetic industries. We show that KA greatly lowers minimum inhibitory (MIC) or fungicidal (MFC) concentrations of commercial medicinal and agricultural antifungal agents, amphotericin B (AMB) and strobilurin, respectively, against pathogenic yeasts and filamentous fungi. Assays using two mitogen-activated protein kinase (MAPK) mutants, i.e., sakAΔ, mpkCΔ, of Aspergillus fumigatus, an agent for human invasive aspergillosis, with hydrogen peroxide (H2O2) or AMB indicate such chemosensitizing activity of KA is most conceivably through disruption of fungal antioxidation systems. KA could be developed as a chemosensitizer to enhance efficacy of certain conventional antifungal drugs or fungicides.

Kojic acid (KA, Figure 1) is a natural pyrone produced by certain filamentous fungi, mainly species of Aspergillus and Penicillium. It is a common by-product in the fermentation of soy sauce, sake and rice wine, and is widely used as a food additive to prevent oxidative browning, or in cosmetics as a depigmenting agent [1–3]. Genes involved in KA biosynthesis were recently identified [4,5]. Cellular immunity is enhanced by KA through stimulating phagocytosis and generation of reactive oxygen species (ROS) in macrophages, and potentiation of phytohemagglutinin-based proliferation of lymphocytes [6,7]. KA is fungistatic against the pathogenic yeast, Cryptococcus neoformans, by inhibiting melanin production required for infectivity [8]. Derivatives of KA also have antimicrobial activity against a variety of other fungi and bacteria [9], showing its potential as a polyfunctional backbone for new antimicrobial agents [10].

Among Aspergillus species, A. flavus, A. parasiticus and A. oryzae are the main producers of KA [11]. A. oryzae is used widely in the food industry. However, A. flavus and A. parasiticus are opportunistic pathogens of various crops, and a concern since they produce carcinogenic aflatoxins that can contaminate food. A. flavus is also an agent for human invasive aspergillosis (IA). Of note, the chief agent of IA, A. fumigatus, and a third IA agent, A. terreus, do not produce KA [12–14].

Co-application of certain types of compounds can enhance efficacy of conventional antimicrobial agents through a process termed “chemosensitization.” With regard to microbial pathogens, a chemosensitizer functions by debilitating the ability of a pathogen to completely activate a defense mechanism to an antimicrobial agent [15,16]. We investigated if KA, as a chemosensitizer, could improve activity of commercial antifungal agents against pathogenic strains of Aspergillus and yeasts (See Table 1). We tested this chemosensitizing potential by co-applying KA with hydrogen peroxide (H2O2) to mimic host ROS, and with a commercial antimycotic, amphotericin B (AMB) and agricultural fungicides, fludioxonil (FLUD) and strobilurin (kresoxim methyl (Kre-Me)).

Synergistic FICIs and FFCIs between KA and H2O2 only occurred in AF293. Despite the absence of calculated “synergism” as depicted by “indifferent” interactions (by definition) (Table 2), there was enhanced antifungal activity (i.e., chemosensitization) in the remaining A. fumigatus and A. terreus strains. This enhancement was indicated by lower MICs and MFCs for either or both KA and H2O2 when co-applied. Also, the A. fumigatus MAPK mutants had half the MICs and MFCs of AF293 (Table 2; Figure 3a), suggesting that, in the wild type fungi, MAPKs in the oxidative/osmotic stress responsive pathway play protective roles against the antimycotic activity of KA + H2O2.

2.2. Enhanced Antimycotic Activity of AMB with KA in Filamentous Fungi and Yeasts

AMB is an antimycotic drug against filamentous or yeast pathogens. However, AMB can be associated with significant side effects resulting in nephrosis and other tissue-damage in invasive pulmonary aspergillosis [23]. Therefore, we reasoned that use of chemosensitizing agents from natural sources could enhance the effectiveness of AMB, while lowering toxicity of this polyene drug to human cells. The main mode of action of AMB is disruption of the fungal plasma membrane, resulting in ion leakage. However, AMB also induces oxidative damage [24–27] by stimulating ROS production [28]. Since KA contributed to oxidative stress when combined with H2O2 in Aspergillus (See Table 2), we surmised it might also enhance AMB activity.

2.2.1. Microtiter Plate (microdilution) Bioassay: Filamentous Fungi

Checkerboard assays of KA (0.2–12.8 mM) and AMB (0.125–32 μg/mL) (See Experimental Section) were initially used to assess antifungal interactions against the Aspergillus strains, by using CLSI M38-A protocol [20]. In assays of the Aspergillus strains, co-application of KA increased AMB activity only in strains of A. fumigatus, where FICIs and FFCIs were synergistic in the A. fumigatus MAPK mutant strains (Table 3; Figure 3b).

2.2.2. Microtiter Plate (microdilution) Bioassay: Yeasts

Checkerboard assays of the yeast strains employed methods outlined in the European Committee on Antimicrobial Susceptibility Testing (EUCAST)] [29]. According to these methods, MICs were determined at 24 h for Candida and Saccharomyces, and at 48 h for Cryptococcus. Following MIC determinations, MFCs were determined on Yeast Peptone Dextrose (YPD) agar, where cells were cultured for an additional 48 h for Candida/Saccharomyces or 72 h for Cryptococcus, respectively.

We also tested combinations of KA with agricultural fungicides, fludioxonil (FLUD) or Kre-Me (strobilurin), fungicides that target different components of the oxidative stress response system [33,34], by using A. fumigatus wild type and MAPK (sakAΔ, mpkCΔ) mutants. Certain fungi with mutations in genes involved in signal transduction of stress response, e.g., MAPK signaling pathway, can escape toxicity of the commercial fungicide FLUD [34]. In a prior study we found redox-active benzo derivatives co-applied with either of these fungicides reduced effective dosages and prevented tolerance of A. fumigatus sakAΔ and mpkCΔ mutants to FLUD [35]. However, in our present study, co-application of KA with FLUD did not overcome tolerance of these mutants to this fungicide (Figure 4a).

In a parallel study, we tested combinations of KA with Kre-Me. Kre-Me is an inhibitor of complex III of the mitochondrial respiratory chain (MRC), the key route system for cellular energy (ATP) production [36]. Moreover, disruption of complex III of the MRC results in an abnormal release of electrons that additionally cause cellular oxidative stress [37]. Therefore, antioxidant enzymes play important roles in protecting cells from oxidative damage triggered by MRC inhibitors. KA improved antimycotic activity of Kre-Me against all A. fumigatus strains (Figure 4b), where A. fumigatus sakAΔ and mpkCΔ mutants showed relatively higher tolerance to Kre-Me than the wild type (AF293). Thus, results indicated that the chemosensitizing mechanism of KA might not involve glutathione/superoxide dismutase-based oxidative stress response, differing from redox-active benzo derivatives [35]. We speculated that, in addition to inhibiting ATP production, co-application of KA and Kre-Me might involve responses of other types of antioxidant enzymes/systems. Comprehensive chemosensitization tests using KA with additional strobilurins are currently underway in various filamentous fungi, including Aspergillus, Penicillium, Acremonium, Scedosporium, and others (Note: There was no chemosensitization effect of KA with any azole drug, such as fluconazole, ketoconazole, itraconazole, in Aspergillus or yeasts (data not shown)).

In the plate bioassay, measurement of sensitivities of filamentous fungi to the antifungal agents was based on percent (%) radial growth of treated compared to control (“No treatment”) fungal colonies (See text for test concentrations.) [38]. Minimum inhibitory concentration (MIC) values on agar plates were determined based on triplicate bioassays, and defined as the lowest concentration of agents where no fungal growth was visible on the plate. For the above assays, fungal conidia (5 × 104 CFU/mL) were diluted in phosphate-buffered saline (PBS) and applied as a drop onto the center of PDA plates with or without antifungal compounds. Growth was observed for three to seven days to determine cellular sensitivities to drugs/compounds.

In summary, enhancing antifungal interactions of KA in combination with H2O2, AMB, FLUD or Kre-Me were, as follows: (1) All A. fumigatus strains were sensitive to either KA + H2O2 or KA + AMB; (2) A. terreus strains were only sensitive to KA + H2O2; (3) C. albicans CAN276, C. krusei ATCC6258, C. neoformans CN24, S. cerevisiae were only sensitive to KA + AMB; and (4) A. flavus 3357, A. parasiticus 5862, C. albicans 90028, C. krusei CAN75, C. tropicalis CAN286 were marginally or not sensitive to any co-treatments; (5) A. fumigatus AF293 was more sensitive than the MAPK mutant strains to KA + Kre-Me. Thus, the antifungal chemosensitizing capacity of KA appears to be antifungal agent and/or fungal strain-specific. In conclusion, KA, a safe natural compound, may have a new use as an enhancer of certain commercial antifungal agents, such as AMB, H2O2 or strobilurin, against defined fungal pathogens. The enhancing effect appears to involve the modulation of the function of oxidative stress response system in the fungus. Further studies are warranted to determine the precise mechanism of action of KA for antifungal chemosensitization.

Acknowledgments

We thank Gregory S. May at The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA, for providing Aspergillus fumigatus (AF293, sakAΔ and mpkCΔ mutants) strains and Arun Balajee, Centers for Disease Control and Prevention, Atlanta, GA, USA, for the strains of A. terreus. This research was conducted under USDA-ARS CRIS Project 5325-42000-037-00D.

Kojic acid was tested up to 12.8 mM. For calculation purpose, 25.6 mM (doubling of 12.8 mM) was used.

Table 3

Antifungal chemosensitization of kojic acid (mM) with AMB (μg/mL) tested against Aspergillus and yeast strains. a MFCs are concentrations where ≥99.9% fungal death was achieved, except where noted in the Table.